Cloning and Expression Analysis of F3'5'H Gene in Three Colors of Scutellaria baicalensis
Flavonoid 3',5'-hydroxylase(F3'5'H)is a key enzyme catalyzing the formation of blue del-phinidin in the anthocyanin biosynthesis pathway.The full-length sequence of F3'5'H genes were cloned from Scutellaria baicalensis with bluish-purple,rose-red and white flowers using RT-PCR method,and they were named as SbF3'5'H-b,SbF3'5'H-r and SbF3'5'H-w,respectively.Sequence alignment showed that compared with SbF3'5'H-b/w,SbF3'5'H-r gene owned 7-bp deficient resulting in early termination of protein transla-tion.Bioinformatics analysis revealed that all the three SbF3'5'H proteins belonged to the cytochrome P450 gene family and were located in the endoplasmic reticulum.SbF3'5'H-B/W protein had a typical CYP450 con-served sequence,including proline-rich motif'LPPGP',substrate recognition site SRS1-6,fold-lock motif'E××R',heme-binding motif'PFGAGRRICAG',and predicted hydroxylation active site CR1 and CR2.However,the sequence deletion of SbF3'5'H-R protein affected its secondary and tertiary structures,and lea-ded to incomplete CYP450 domain,lacking several conserved motifs that maintain enzyme activity at C termi-nus.The phylogenetic evolution based on F3'5'H protein sequence indicated that S.baicalensis was closely re-lated to Salvia miltiorrhiza and Salvia splendens,which also belonged to Labiaceae family and were clustered in one branch,while was far related to monocotyledon plants.Expression analysis showed that the expression lev-el of SbF3'5'H gene was the highest in white flowers,followed by bluish-purple flowers,and was the lowest in rose-red flowers.Comparing among different flower developmental stages,SbF3'5'H gene showed the trend of increasing firstly and then decreasing,and had the highest expression level at the middle bud stage,followed by the young bud stage,and had the lowest level at the full bloom stage.There were significant or extremely significant differences among different stages of flower development in bluish-purple and white flowers.These results would provide important guidance for further research on the functional differences of SbF3'5'H genes in S.baicalensis with different flower colors and revealing the molecular mechanism of color variation.
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